Regarding biosynthesis of human highly unsaturated fatty acids (hereinafter referred to as “PUFA”), there are two typical series, ω3 and ω6 series. ω (omega) refers to the number of carbon atoms from the terminal methyl group of the fatty acid to the carbon atom at which the first double bond is located. For example, in the case of the ω6 series, linoleic acid (18:2 ω6) is repeatedly subjected to unsaturation and carbon chain length extension and consequently converted to γ-linolenic acid (18:3 ω6), dihomo-γ-linolenic acid (20:3 ω6), arachidonic acid (20:4 ω6), and 4,7,10,13,16-docosapentaenoic acid (22:5 ω6).
Likewise, in the case of the ω3 series, α-linolenic acid (18:3 ω3) is repeatedly subjected to unsaturation and carbon chain length extension and consequently is converted to eicosapentaenoic acid (20:5 ω3), 7,10,13,16,19-docosapentaenoic acid (22:5 ω3) and 4,7,10,13,16,19-docosahexaenoic acid (22:6 ω3). Eicosapentaenoic acid (hereinafter referred to as “EPA” and docosahexaenoic acid (hereinafter referred to as “DHA”) as PUFAs of the ω3 series are known to have many physiological functions, particularly prophylactic effects for lifestyle diseases, such as arterial sclerosis and thrombosis, and carcinostatic action and learning ability enhancing action, and various attempts have been made to apply these PUFAs to pharmaceutical preparations and specific food products for health care. In recent years, however, attention has also been drawn to physiological functions of PUFAs other than the ω3 series ω6 and ω9 series).
About 10% of fatty acids constituting important organs such as blood and liver is accounted for by arachidonic acid. For example, fatty acids in phospholipids of human blood have a composition comprising 11% of arachidonic acid, 1% of eicosapentaenoic acid, and 3% of docosahexaenoic acid. The arachidonic acid is involved, as a major constituent component of a cell membrane, in the regulation of flow in the membrane to exhibit various functions in metabolism in the body and, further, plays an important role as a direct precursor of prostaglandins.
In particular, the arachidonic acid has recently drawn attention as a nutrient for infants and as a constituent fatty acid of endogenous cannabinoid (2-arachidonoyl monoglycerol, anandamide) having a nerve activating action. In general, upon ingestion of a food rich in linoleic acid, linoleic acid is converted to arachidonic acid. In patients suffering from lifestyle diseases and a reserve group of lifestyle diseases, infants, and aged persons, however, the function of enzyme involved in the biosynthesis is lowered. Therefore, the amount of arachidonic acid is likely to be deficient. For this reason, it is desirable to ingest the arachidonic acid directly as a fat or oil (constituent fatty acid of triglyceride).
For EPA and DHA as PUFAs of the ω3 series, there is fish oil as an abundant supply source. On the other hand, γ-linolenic acid, dihomo-γ-linoleinic acid, arachidonic acid, and 4,7,10,13,16-docosapentaenoic acid (22:5 ω6) as PUFAs of ω6 series are hardly obtained from conventional fat or oil supply sources, and, at the present time, fat or oil (triglycerides) comprising, as a constituent fatty acid, PUFAs produced by fermentation of microorganisms are generally used. For example, a method has been proposed wherein a fat or oil (triglyceride) comprising arachidonic acid as a constituent fatty acid is produced by culturing various microorganisms capable of producing fat or oil (triglycerides) comprising arachidonic acid as a constituent fatty acid.
Among others, the production of fat or oil containing a high arachidonic acid content (triglycerides) by using particularly microorganisms belonging to the genus Mortierella is known (Japanese Unexamined Patent Publication (Kokai) Nos. 63 (1988)-44891 and 63 (1988)-12290). Based on many test results, these fat or oil are said to be safe. As, however, this fat or oil is derived from microorganisms, there is no satisfactory experiences in ingestion. Therefore, at the present time, the fat or oil in question have not yet satisfactorily infiltrated into the society. On the other hand, fat or oil (triglycerides) comprising, as a constituent fatty acid, arachidonic acid produced by fermentation have begun to be used in applications where arachidonic acid should be used, for example, in the field of infant nutrition and, specifically, in infant formula.
The fat or oil produced from naturally occurring products such as animals and plants are subjected to refining processes, such as degumming, deoxidation, deodorization, decoloration, molecular distillation, and wintering and are then put on the market as edible fat or oil. For example, fat or oil obtained by squeezing from oil plants contain a large amount of impurities and thus as such cannot be used as edible fat or oil. Except for sesame oils and olive oils which have often been eaten, these fat or oil are generally refined before use as edible oils.
For example, in the degumming process, phospholipids, carbohydrates, resins, protein compounds, trace metals, and coloring matter contained in unrefined oils are removed. In the deoxidation (alkali refining) process, fatty acids, coloring matter, phospholipids, trace metals, sulfur compounds, oil insolubles, and oxidation products are removed. In the decoloration process, coloring matter, gummy matter, trace metals, soap components, oxidation products, and phospholipids are removed. In the deodorization process, fatty acids, monoglycerides, diglycerides, aldehydes, alcohols, ketones, hydrocarbons, coloring matter, sulfur compounds, peroxides, oxidative degradation products and other odor components are removed.
Fat or oil contain organic compounds, which are soluble in oils and are less likely to be degraded with an alkali, called “unsaponifiable matter.” For example, compounds such as higher alcohols, sterols, hydrocarbons, tocopherols and carotenoids are known as constituent components of unsaponifiable matter. In the fat or oil refining processes, unsaponifiable matter contents can be reduced but cannot be fully removed. Sterols are known to exist as main components of unsaponifiable matter in fat or oil produced by microorganisms.
Sterols present in fat or oil are divided into free types and ester types. In the refining processes, the free type can be removed, but on the other hand, the ester type can hardly be removed. For example, after degumming, deoxidation, decoloration, and deodorization processes, the contents of the sterol content (mg/g) of soybean oil are 3.4, 3.0, 2.0, and 1.6, respectively, for the free type, and is 0.6, 0.6, 0.6, and 0.6, respectively, for the ester type (“Shokuyo Yushi no Kagaku (Science of edible fat or oil),” pp. 20-21, SAIWAISHOBO).
As irremovable sterols and the like are contained as a part of unsaponifiable matter, in general, the unsaponifiable matter content is extensively used as an index of the quality of refined fat or oil or as a control index of the refining process. For example, according to Japanese Agricultural Standards, the content of unsaponifiable matter, e.g., in edible safflower oils, edible soybean oils, and edible palm oils should be not more than 1.0% (the 523rd notification (Mar. 31, 1969) of the Ministry of Agriculture, Forestry and Fisheries). For infants, cholesterol is necessary, and an infant formula having an increased cholesterol content is on the market. Plant-derived sterols are contained in vegetable edible fat or oil. The presence of the vegetable-derived sterols disadvantageously inhibits cholesterol absorption in infants (Shokuhin to Kaihatsu (Food Processing And Ingredients), Vol. 33, No. 2, pp. 42-45 (1998)). Therefore, when applications where edible fat or oil are incorporated in infant formula are taken into consideration, edible fat or oil having low sterol content, that is, having a low unsaponifiable matter content, are strongly desired.
The major part of fat or oil produced by culturing microorganisms belonging to the genus Mortierella are accounted for by triglycerides (not less than about 70% by weight) and phospholipids, and unsaponifiable matter is contained as other component. The unsaponifiable matter comprises sterols, such as desmosterol, and sterol esters as main components. Edible fat or oil are in the form of triglycerides. Refined fat or oil, from which phospholipids have been removed, can be provided by subjecting original fat or oil produced by culturing microorganisms (fat or oil which have been provided by extraction of cells and are called “crude oil”) to refining processes for edible fat or oil (degumming, deoxidation, deodorization, and decoloration). However, it is difficult to fully remove unsaponifiable matter by the refining processes.
For the reasons that full removal of the unsaponifiable matter is difficult and edible fat or oil having low unsaponifiable matter content are widely desired, studies have been made on how to remove unsaponifiable matter. As a result, refining by column chromatography has been developed (Japanese unexamined Patent Publication (Kokai) No. 10 (1998)-191886). In this technique, however, no detailed studies have been made on components constituting the unsaponifiable matter. A change in components of unsaponifiable matter after refining from the components before the refining, that is, information on which components have been removed by refining, remains unknown.
Fat or oil produced by culturing microorganisms belonging to the genus Mortierella are accumulated within mycelia. Therefore, culture should be carried out to give higher cell concentration from the viewpoint of improving the cost effectiveness of the production of highly unsaturated fatty acid-containing fat or oil. In order to provide a high cell concentration, the concentration of nitrogen source of the medium converted to cell components should be increased. According to previous reports (Japanese Unexamined Patent Publications (Kokai) No. 10 (1998)-191886 and No. 10 (1998)-70992), although the unsaponifiable matter or total sterol content is reported, the concentration of the nitrogen source in the medium used in culture of microorganisms is about 1.5% at the highest.
Further, highly unsaturated fatty acid-containing fat or oil having a sterol content of not more than 1% have been reported (Published Japanese Translation of PCT Publication No. 2000-510513). In this case, however, the concentration of a nitrogen source in a medium used in this production is low, and, in the production of a arachidonic acid-containing fat or oil produced using Mortierella alpina, culture is carried out in a low nitrogen source concentration of 1% (=yeast extract 0.5%+NaNO3 0.5%) (Published Japanese Translation of PCT Publication No. 2000-508888). Thus, for the reason that edible fat or oil having low unsaponifiable matter content and sterol content are extensively desired, studies have been made on a reduction in unsaponifiable matter and/or sterols. However, there is no description on unsaponifiable matter and sterols contained in microbial fat or oil produced by high-concentration culture of microorganisms.
As described above, sterols as a main component of unsaponifiable matter are divided into free-type sterols and ester-type sterols. The free-type sterols can be removed by the refining processes, whereas the ester-type sterols can be hardly removed by the refining processes. Therefore, it is considered that, in order to provide refined fat or oil having low unsaponifiable matter content, specifically low sterol contents, the development of a raw material for refining having a low content of ester-type sterols, which cannot be removed by the refining processes without difficulties, that is, a crude oil having low ester type-sterol content, is important.